www.nature.com/scientificreports
OPEN
received: 28 September 2016 accepted: 02 December 2016 Published: 10 January 2017
Rye polyphenols and the metabolism of n-3 fatty acids in rats: a dose dependent fatty fish-like effect Fayçal Ounnas1,2, Michel de Lorgeril1, Patricia Salen1, François Laporte3, Luca Calani4, Pedro Mena4, Furio Brighenti4, Daniele Del Rio4 & Christine Demeilliers2 As long-chain fatty acids (LCFA) of the n-3 series are critically important for human health, fish consumption has considerably increased in recent decades, resulting in overfishing to respond to the worldwide demand, to an extent that is not sustainable for consumers’ health, fisheries economy, and marine ecology. In a recent study, it has been shown that whole rye (WR) consumption improves blood and liver n-3 LCFA levels and gut microbiota composition in rats compared to refined rye. The present work demonstrates that specific colonic polyphenol metabolites may dose dependently stimulate the synthesis of n-3 LCFA, possibly through their microbial and hepatic metabolites in rats. The intake of plant n-3 alpha-linolenic acid and WR results in a sort of fatty fish-like effect, demonstrating that the n-3 LCFA levels in blood and tissues could be increased without eating marine foods, and therefore without promoting unsustainable overfishing, and without damaging marine ecology. Long-chain fatty acids (LCFA) of the n-3 series — also called marine LCFA because marine foods are the main source of these lipids — are critically important for human health1. Fish consumption has considerably increased in recent decades, resulting in overfishing to respond to the worldwide demand2, and, at the same time, sea and ocean pollution have also increased over the planet3. As a consequence, it is more and more difficult for consumers to obtain high-quality marine foods at reasonable prices2. The present situation is not sustainable for consumers’ health, fisheries’ economy, and marine ecology. We need alternative sources of marine n-3 LCFA, and this made fish farming (or pisciculture) grow relevantly. However, farming carnivorous fish (e.g. salmon) does not reduce pressure on wild fisheries, since they require fishmeal extracted from wild forage fish. On the other hand, the amounts of marine n-3 LCFA in herbivorous fishes (e.g. carp and tilapia) are quite low and will not be able to meet the future growing demand. Seaweed farming may be another solution, and marine n-3 LCFA have now been produced in large-scale cultivation of microalgae, but this is unlikely to become an affordable reality in the short-term, particularly because of high production costs, quite limited yields in marine n-3 LCFA, and consumer reluctance. Finally, marine n-3 LCFA could also be produced from terrestrial plants via transgenic means, which will eventually clear regulatory hurdles for commercialisation, but societal acceptance remains in question. For the present time, no affordable, environmentally sustainable, and socially acceptable solution exists. Since humans have the ability to synthesize marine n-3 LCFA from plant n-3 alpha-linolenic acid (although insufficiently), one solution would be to stimulate the endogenous production of n-3 LCFA by the human body. For instance, certain polyphenols have been shown able to increase the concentrations of marine n-3 LCFA in animals4,5 and humans6,7. In a recent study, it has been shown that whole rye (WR) consumption improves blood and liver n-3 LCFA levels and gut microbiota composition in rats compared to refined rye8. This might be partly
1
Laboratoire TIMC-IMAG CNRS UMR 5525, Cœur et Nutrition, Université Grenoble-Alpes, Grenoble, France. Laboratory of Fundamental and Applied Bioenergetics, Environmental and Systems Biology, Inserm, U1055, Université Grenoble-Alpes, Grenoble France. 3Département de Biochimie, Pharmacologie et Toxicologie, Unité Biochimie Hormonale et Nutritionnelle, Centre Hospitalier et Universitaire de Grenoble, France. 4Laboratory of Phytochemicals in Physiology, Department of Food Science, University of Parma, Medical School, Building C, Parma, Italy. Correspondence and requests for materials should be addressed to D.D.R. (email: [email protected]) 2
Scientific Reports | 7:40162 | DOI: 10.1038/srep40162
1
www.nature.com/scientificreports/ involved in the health benefits associated with WR consumption, particularly a lower risk of metabolic syndrome and cancer9,10. However, the totality of mechanisms by which WR is beneficial is not fully understood. Also, the specific substances potentially implicated in WR health benefits — and present in the outer part of the rye grain — have not been fully identified. As an example, WR contains several polyphenols, including lignans and phenolic acids, which can influence the endogenous biosynthesis of n-3 LCFA. Rye lignans are converted by gut bacteria into enterodiol and enterolactone11,12 that possess hormonal activity, and this may partly modulate the synthesis of n-3 LCFA in humans13–15. In addition, WR consumption in rats results in increased urinary excretion of phenolic acid metabolites, which may also influence the metabolism of n-3 LCFA5,8,16. In the above-cited animal study comparing refined rye and WR, we did not evaluate the dose–effect relationship — a useful approach to demonstrate a causal effect — between rye polyphenols and n-3 LCFA, and whether specific rye polyphenol metabolites were involved in the metabolism n-3 LCFA8. Therefore, the present study was designed to confirm the effect of rye polyphenols on the endogenous metabolism of n-3 LCFA in rats, in order to examine a dose–effect relationship and to identify which polyphenol metabolites are potentially involved in the process. We compared three groups of rats, where two groups were fed two different doses of WR, whereas a control group did not consume rye. We used a multiple linear regression model to analyse the associations between urinary rye polyphenol metabolites and systemic and hepatic marine n-3 LCFA.
Materials and Methods
Animals and experimental diets. Thirty-six male Wistar rats (Charles River Laboratories, l′Arbresle, France, baseline body weight 75–100 g) were fed a standard diet (A04, SAFE Diets, France). They were housed in individual cages under conditions of constant temperature and humidity and a standard light-dark cycle (12 h/12 h). Tap water and standard diet were provided ad libitum. The standard diet was A04, from SAFE Diets (France) and did not contain any WR (control diet, CT). The experimental diets were prepared by mixing the same standard diet A04 flour with either 39% WR (WR39) or 79% WR (WR79) as a replacement for A04 cereals. The amounts of nutrients provided by the two experimental diets were adjusted to the recommendations of the American Institute of Nutrition Rodent Diets-9317. Experimental design. The rats were cared for in accordance with the European Council Directive 86/609/ EEC on the care and use of laboratory animals (OJL 358). Protocols were carried out under license from the French Ministry of Agriculture (Permit N° A380727) and approved by the Committee on the Ethics of Animal Experiments of the University of Grenoble (Permit N° 113_ LBFA-FO-01). All efforts were made to minimise animal suffering. The animals were acclimatised one week before being randomly distributed into three groups (n = 12/group). The rats were then fed either the CT or the WR39 or the WR79 diets during 12 weeks. Weight and food consumption were recorded weekly. One week before sacrifice, urine was sampled in individual metabolic cages. At the end of the experiment, plasma and liver were sampled. All samples were immediately frozen in liquid nitrogen and stored at −80 °C until analysis. Analysis of polyphenolic compounds in plasma and urine. Urine samples were diluted with 0.1% aqueous formic acid and filtered through a 0.45 μm nylon filter prior to UHPLC-MSn (LTQ XL, Thermo Fisher Scientific Inc., San Jose, CA, USA) as previously reported8. Fatty acid analyses. Plasma and liver lipids were extracted in hexane/isopropanol and quantitated as previously described6,7. Briefly, methylated fatty acids were extracted with hexane, separated, and quantified by GC using a 6850 Series gas chromatograph system (Agilent Technologies, Palo Alto, CA, USA). Plasma is the main biological factor involved in fatty acid distribution in the body and the liver is regarded the main source of endogenous synthesis of polyunsaturated fatty acids in mammals. We focused our analyses on the changes in n-6 and n-3 fatty acids, in particular eicosapentanoic acid (EPA, 20:5n-3) and docosahexanoic acid (DHA, 22:6n-3). Statistical analysis. Between-group differences in physiological characteristics, urinary polyphenols and
blood and liver fatty acids were evaluated using analysis of variance, and individual group differences were evaluated when necessary with post-hoc Fisher’s LSD test (significance p 0.05) one-way ANOVA. *Indicate statistical difference between the groups: *p
OPEN
received: 28 September 2016 accepted: 02 December 2016 Published: 10 January 2017
Rye polyphenols and the metabolism of n-3 fatty acids in rats: a dose dependent fatty fish-like effect Fayçal Ounnas1,2, Michel de Lorgeril1, Patricia Salen1, François Laporte3, Luca Calani4, Pedro Mena4, Furio Brighenti4, Daniele Del Rio4 & Christine Demeilliers2 As long-chain fatty acids (LCFA) of the n-3 series are critically important for human health, fish consumption has considerably increased in recent decades, resulting in overfishing to respond to the worldwide demand, to an extent that is not sustainable for consumers’ health, fisheries economy, and marine ecology. In a recent study, it has been shown that whole rye (WR) consumption improves blood and liver n-3 LCFA levels and gut microbiota composition in rats compared to refined rye. The present work demonstrates that specific colonic polyphenol metabolites may dose dependently stimulate the synthesis of n-3 LCFA, possibly through their microbial and hepatic metabolites in rats. The intake of plant n-3 alpha-linolenic acid and WR results in a sort of fatty fish-like effect, demonstrating that the n-3 LCFA levels in blood and tissues could be increased without eating marine foods, and therefore without promoting unsustainable overfishing, and without damaging marine ecology. Long-chain fatty acids (LCFA) of the n-3 series — also called marine LCFA because marine foods are the main source of these lipids — are critically important for human health1. Fish consumption has considerably increased in recent decades, resulting in overfishing to respond to the worldwide demand2, and, at the same time, sea and ocean pollution have also increased over the planet3. As a consequence, it is more and more difficult for consumers to obtain high-quality marine foods at reasonable prices2. The present situation is not sustainable for consumers’ health, fisheries’ economy, and marine ecology. We need alternative sources of marine n-3 LCFA, and this made fish farming (or pisciculture) grow relevantly. However, farming carnivorous fish (e.g. salmon) does not reduce pressure on wild fisheries, since they require fishmeal extracted from wild forage fish. On the other hand, the amounts of marine n-3 LCFA in herbivorous fishes (e.g. carp and tilapia) are quite low and will not be able to meet the future growing demand. Seaweed farming may be another solution, and marine n-3 LCFA have now been produced in large-scale cultivation of microalgae, but this is unlikely to become an affordable reality in the short-term, particularly because of high production costs, quite limited yields in marine n-3 LCFA, and consumer reluctance. Finally, marine n-3 LCFA could also be produced from terrestrial plants via transgenic means, which will eventually clear regulatory hurdles for commercialisation, but societal acceptance remains in question. For the present time, no affordable, environmentally sustainable, and socially acceptable solution exists. Since humans have the ability to synthesize marine n-3 LCFA from plant n-3 alpha-linolenic acid (although insufficiently), one solution would be to stimulate the endogenous production of n-3 LCFA by the human body. For instance, certain polyphenols have been shown able to increase the concentrations of marine n-3 LCFA in animals4,5 and humans6,7. In a recent study, it has been shown that whole rye (WR) consumption improves blood and liver n-3 LCFA levels and gut microbiota composition in rats compared to refined rye8. This might be partly
1
Laboratoire TIMC-IMAG CNRS UMR 5525, Cœur et Nutrition, Université Grenoble-Alpes, Grenoble, France. Laboratory of Fundamental and Applied Bioenergetics, Environmental and Systems Biology, Inserm, U1055, Université Grenoble-Alpes, Grenoble France. 3Département de Biochimie, Pharmacologie et Toxicologie, Unité Biochimie Hormonale et Nutritionnelle, Centre Hospitalier et Universitaire de Grenoble, France. 4Laboratory of Phytochemicals in Physiology, Department of Food Science, University of Parma, Medical School, Building C, Parma, Italy. Correspondence and requests for materials should be addressed to D.D.R. (email: [email protected]) 2
Scientific Reports | 7:40162 | DOI: 10.1038/srep40162
1
www.nature.com/scientificreports/ involved in the health benefits associated with WR consumption, particularly a lower risk of metabolic syndrome and cancer9,10. However, the totality of mechanisms by which WR is beneficial is not fully understood. Also, the specific substances potentially implicated in WR health benefits — and present in the outer part of the rye grain — have not been fully identified. As an example, WR contains several polyphenols, including lignans and phenolic acids, which can influence the endogenous biosynthesis of n-3 LCFA. Rye lignans are converted by gut bacteria into enterodiol and enterolactone11,12 that possess hormonal activity, and this may partly modulate the synthesis of n-3 LCFA in humans13–15. In addition, WR consumption in rats results in increased urinary excretion of phenolic acid metabolites, which may also influence the metabolism of n-3 LCFA5,8,16. In the above-cited animal study comparing refined rye and WR, we did not evaluate the dose–effect relationship — a useful approach to demonstrate a causal effect — between rye polyphenols and n-3 LCFA, and whether specific rye polyphenol metabolites were involved in the metabolism n-3 LCFA8. Therefore, the present study was designed to confirm the effect of rye polyphenols on the endogenous metabolism of n-3 LCFA in rats, in order to examine a dose–effect relationship and to identify which polyphenol metabolites are potentially involved in the process. We compared three groups of rats, where two groups were fed two different doses of WR, whereas a control group did not consume rye. We used a multiple linear regression model to analyse the associations between urinary rye polyphenol metabolites and systemic and hepatic marine n-3 LCFA.
Materials and Methods
Animals and experimental diets. Thirty-six male Wistar rats (Charles River Laboratories, l′Arbresle, France, baseline body weight 75–100 g) were fed a standard diet (A04, SAFE Diets, France). They were housed in individual cages under conditions of constant temperature and humidity and a standard light-dark cycle (12 h/12 h). Tap water and standard diet were provided ad libitum. The standard diet was A04, from SAFE Diets (France) and did not contain any WR (control diet, CT). The experimental diets were prepared by mixing the same standard diet A04 flour with either 39% WR (WR39) or 79% WR (WR79) as a replacement for A04 cereals. The amounts of nutrients provided by the two experimental diets were adjusted to the recommendations of the American Institute of Nutrition Rodent Diets-9317. Experimental design. The rats were cared for in accordance with the European Council Directive 86/609/ EEC on the care and use of laboratory animals (OJL 358). Protocols were carried out under license from the French Ministry of Agriculture (Permit N° A380727) and approved by the Committee on the Ethics of Animal Experiments of the University of Grenoble (Permit N° 113_ LBFA-FO-01). All efforts were made to minimise animal suffering. The animals were acclimatised one week before being randomly distributed into three groups (n = 12/group). The rats were then fed either the CT or the WR39 or the WR79 diets during 12 weeks. Weight and food consumption were recorded weekly. One week before sacrifice, urine was sampled in individual metabolic cages. At the end of the experiment, plasma and liver were sampled. All samples were immediately frozen in liquid nitrogen and stored at −80 °C until analysis. Analysis of polyphenolic compounds in plasma and urine. Urine samples were diluted with 0.1% aqueous formic acid and filtered through a 0.45 μm nylon filter prior to UHPLC-MSn (LTQ XL, Thermo Fisher Scientific Inc., San Jose, CA, USA) as previously reported8. Fatty acid analyses. Plasma and liver lipids were extracted in hexane/isopropanol and quantitated as previously described6,7. Briefly, methylated fatty acids were extracted with hexane, separated, and quantified by GC using a 6850 Series gas chromatograph system (Agilent Technologies, Palo Alto, CA, USA). Plasma is the main biological factor involved in fatty acid distribution in the body and the liver is regarded the main source of endogenous synthesis of polyunsaturated fatty acids in mammals. We focused our analyses on the changes in n-6 and n-3 fatty acids, in particular eicosapentanoic acid (EPA, 20:5n-3) and docosahexanoic acid (DHA, 22:6n-3). Statistical analysis. Between-group differences in physiological characteristics, urinary polyphenols and
blood and liver fatty acids were evaluated using analysis of variance, and individual group differences were evaluated when necessary with post-hoc Fisher’s LSD test (significance p 0.05) one-way ANOVA. *Indicate statistical difference between the groups: *p
